To initiate its life cycle, human papillomavirus (“HPV”) needs to enter the basal keratinocyte, the only dividing cell in the normal stratified epithelium, which is able to provide HPV with the necessary DNA replicative molecular machinery it lacks. Viral entry occurs through at least one receptor that was initially believed to be an alpha-6 integrin. However, subsequent work failed to confirm the observation (Evander et al., “Identification of the Alpha6 Integrin as a Candidate Receptor for Papillomaviruses,” J. Virol. 71(3):2449-2456 (1997) and Giroglou et al., “Human Papillomavirus Infection Requires Cell Surface Heparan Sulfate,” J. Virol. 75(3):1565-(570 (2001)). Instead, heparan sulfate, a glycosaminoglycan (GAG), is now regarded as the primary receptor for HPV (Giroglou et al., “Human Papillomavirus Infection Requires Cell Surface Heparan Sulfate,” J. Virol. 75(3):1565-1570 (2001); Joyce et al., “The L1 Major Capsid Protein of Human Papillomavirus Type 11 Recombinant Virus-Like Particles Interacts with Heparin and Cell-Surface Glycosaminoglycans on Human Keratinocytes,”J. Biol. Chem. 274(9):5810-5822 (1999); Combita et al., “Gene Transfer Using Human Papillomavirus Pseudovirions Varies According to Virus Genotype and Requires Cell Surface Heparan Sulfate,” FEMS Microbiol. Lett. 204(1):183-188 (2001); Bousarghin et al., “Positively Charged Sequences of Human Papillomavirus Type 16 Capsid Proteins are Sufficient to Mediate Gene Transfer into Target Cells via the Heparan Sulfate Receptor,” J. Gen. Virol. 84(Pt 1):157-164 (2003); Drobni et al., “Carboxy-Fluorescein Diacetate, Succinimidyl Ester Labeled Papillomavirus Virus-Like Particles Fluoresce after Internalization and Interact with Heparan Sulfate for Binding and Entry,” Virol. 310(1):163-172 (2003); Sapp et al., “Structure. Attachment and Entry of Polyoma- and Papillomaviruses,” Virol. 384(2):400-409 (2009); and Sapp et al., “Viral Entry Mechanisms: Human Papillomavirus and a Long Journey from Extracellular Matrix to the Nucleus,” FEBS J. 276(24):7206-7216 (2009)). This initial binding appears to induce a change in the capsid conformation, followed by the binding to a putative second receptor that leads to cellular entry of the virus. Different mechanisms have been proposed for the virus endocytosis, some involving a clathrin- and caveolin-independent mechanism, but others involving a clathrin-coated pit and caveolae mediated fusion to the endosome (Giroglou et al., “Human Papillomavirus Infection Requires Cell Surface Heparan Sulfate,” J. Virol. 75(3):1565-1570 (2001); Sapp et al., “Structure, Attachment and Entry of Polyoma- and Papillomaviruses,” Virol. 384(2):400-409 (2009); Sapp et al., “Viral Entry Mechanisms: Human Papillomavirus and a Long Journey from Extracellular Matrix to the Nucleus,” FEBS J. 276(24):7206-7216 (2009); Selinka et al., “Analysis of the Infectious Entry Pathway of Human Papillomavirus Type 33 Pseudovirions,” Virol. 299(2):279-287 (2002); and Hindmarsh et al., “Mechanisms Regulating Expression of the HPV 31 L1 and L2 Capsid Proteins and Pseudovirion Entry,” J. Virol. 4:19 (2007)). The second receptor might be an uncharacterized extracellular matrix component, possibly related to laminin (Culp et al., “Human Papillomaviruses Bind a Basal Extracellular Matrix Component Secreted by Keratinocytes which is Distinct from a Membrane-Associated Receptor,” Virol. 347(1):147-159 (2006)).
As a GAG, heparan sulfate represents the polysaccharide moiety that is covalently bound to a protein to form a proteoglycan. Syndecan 1, glypican 1, and syndecan 4 are some of the protein moieties to which heparin sulfate binds, and these proteins contribute to different cellular affinities for the HPV capsids (Shafti-Keramat et al., “Different Heparin Sulfate Proteoglycans Serve as Cellular Receptors for Human Papillomaviruses,” J. Virol. 77(24):13125-13135 (2003)). Heparan sulfate mediates interaction not just with papillomavirus capsids, but also with a wide range of growth factors, morphogens, and receptors. The degree of sulfation (N- and O-sulfation) of heparan sulfate contributes greatly to the various specificities of proteoglycans (Sapp et al., “Structure, Attachment and Entry of Polyoma- and Papillomaviruses,” Virol. 384(2):400-409 (2009); Sapp et al., “Viral Entry Mechanisms: Human Papillomavirus and a Long Journey from Extracellular Matrix to the Nucleus,” FEBS J. 276(24):7206-7216 (2009); Whitelock et al., “Heparan Sulfate: A Complex Polymer Charged with Biological Activity,” Chem. Rev. 105(7):2745-64 (2005); and Kreuger et al., “Interactions Between Heparan Sulfate and Proteins: The Concept of Specificity,” J. Cell Biol. 174(3):323-327(2006)). This initial binding of the HPV virions is thought to occur not on the cell surface, but in the extracellular matrix, around the basement membrane. It is not entirely clear if GAGs are essential for HPV virion entry, as heparan sulfate is not required for infection by HPV-31b virions of a skin organotypic culture (Patterson et al., “Human Papillomavirus Type 31b Infection of Human Keratinocytes does not Require Heparan Sulfate,” J. Virol. 79(11):6838-6847 (2005)). The role of heparans or syndecans in virus cell binding or entry is not limited to papillomavirus; it extends to various herpes viruses, including herpes simplex, adenoviruses and adeno-associated viruses, murine coronavirus, porcine circovirus, dengue, hepatitis C, Sindbis, tick-borne encephalitis, hepatitis B, and HIV (Leistner et al., “Role of Glycosaminoglycans for Binding and Infection of Hepatitis B Virus,” Cell Microbiol. 10(1):122-133 (2008); Perabo et al., “Heparan Sulfate Proteoglycan Binding Properties of Adeno-Associated Virus Retargeting Mutants and Consequences for their in vivo Tropism,” J. Virol. 80(14):7265-7269 (2006); Misinzo et al., “Porcine Circovirus 2 uses Heparan Sulfate and Chondroitin Sulfate B Glycosaminoglycans as Receptors for its Attachment to Host Cells,” J. Virol. 80(7):3487-3494 (2006); de Haan et al., “Murine Coronavirus with an Extended Host Range uses Heparan Sulfate as an Entry Receptor,” J. Virol. 79(22):14451-14456 (2005); Vives et al., “Heparan Sulfate Proteoglycan Mediates the Selective Attachment and Internalization of Serotype 3 Human Adenovirus Dodecahedron,” Virol. 321(2):332-340 (2004); Spear “Herpes Simplex Virus: Receptors and Ligands for Cell Entry,” Cell Microbiol. 6(5):401-410 (2004); Zautner et al., “Heparan Sulfates and Coxsackievirus-Adenovirus Receptor: Each one Mediates Coxsackievirus B3 PD Infection,” J. Virol. 77(18):10071-10077 (2003); Kroschewski et al., “Role of Heparan Sulfate for Attachment and Entry of Tick-Borne Encephalitis Virus,” Virol. 308(1):92-100 (2003); Bobardt et al., “Syndecan Captures, Protects, and Transmits HIV to T Lymphocytes,” Immunity 18(1):27-39 (2003); Shukla et al., “Herpesviruses and Heparan Sulfate: an Intimate Relationship in Aid of Viral Entry,” J. Clin. Invest. 108(4):503-510 (2001); Goodfellow et al., “Echoviruses Bind Heparan Sulfate at the Cell Surface,” J. Virol. 75(10):4918-4921 (2001); Birkmann et al., “Cell Surface Heparin Sulfate is a Receptor for Human Herpesvirus 8 and Interacts with Envelope Glycoprotein K8,” J. Virol. 75(23):11583-11593 (2001); Akula et al., “Human Herpesvirus 8 Interaction with Target Cells Involves Heparan Sulfate,” Virol. 282(2):245-255 (2001); Dechecchi et al., “Heparan Sulfate Glycosaminoglycans are Involved in Adenovirus Type 5 and 2-Host Cell Interactions,” Virol. 268(2):382-390 (2000); Byrnes et al., “Binding of Sindbis Virus to Cell Surface Heparan Sulfate,” J. Virol. 72(9):7349-73456 (1998); Chen et al., “Dengue Virus Infectivity Depends on Envelope Protein Binding to Target Cell Heparan Sulfate,” Nat. Med. 3(8):866-871(1997); Compton et al., “Initiation of Human Cytomegalovirus Infection Requires Initial Interaction with Cell Surface Heparan Sulfate,” Virol. 193(2):834-841 (1993); Spear et al., “Heparan Sulfate Glycosaminoglycans as Primary Cell Surface Receptors for Herpes Simplex Virus,” Adv. Exp. Med. Biol. 313:341-53 (1992); Shieh et al., “Cell Surface Receptors for Herpes Simplex Virus are Heparan Sulfate Proteoglycans,” J. Cell Biol. 116(5):1273-1281 (1992)).
The binding to the heparan sulfate proteoglycan receptor leads to a change in the conformation of the HPV capsid with shifts in the L1 protein followed by shifts in the L2 protein (Sapp et al., “Structure, Attachment and Entry of Polyoma- and Papillomaviruses,” Virol. 384(2):400-409 (2009)). This leads to an exposure of the L2 amino-terminus and its cleavage by furin, a serine endoprotease convertase enzyme that cleaves many precursor proteins. Furin may also contribute to the cellular entry of HIV, as well as to the maturation of the influenza and dengue viruses, among others (Nakayama K., “Furin: A Mammalian Subtilisin/Kex2p-like Endoprotease Involved in Processing of a Wide Variety of Precursor Proteins,” Biochem. J. 327:625-635 (1997); Mukhopadhyay et al., “A Structural Perspective of the Flavivirus Life Cycle,” Nat. Rev. Microbiol. 3(1):13-22 (2005); and Day et al., “The Role of Furin in Papillomavirus Infection,” Future Microbiol. 4:1255-62 (2009)). Cell cyclophilins also appear to facilitate this conformation change and binding to a second entry receptor yet to be identified (Sapp et al., “Structure, Attachment and Entry of Polyoma- and Papillomaviruses,” Virol. 384(2):400-409 (2009); Sapp et al., “Viral Entry Mechanisms: Human Papillomavirus and a Long Journey from Extracellular Matrix to the Nucleus,” FEBS J. 276(24):7206-7216 (2009); and Bienkowska-Haba et al., “Target Cell Cyclophilins Facilitate Human Papillomavirus Type 16 Infection,” PLoS Pathogens 5(7):e1000524 (2009)). This binding then initiates the endocytosis of the virus.
It should be pointed out that different investigators have found differences in the viral entry process according to the kind of HPV type studied (Sapp et al., “Structure, Attachment and Entry of Polyoma- and Papillomaviruses,” Virol. 384(2):400-409 (2009); Sapp et al., “Viral Entry Mechanisms: Human Papillomavirus and a Long Journey from Extracellular Matrix to the Nucleus,” FEBS J. 276(24):7206-7216 (2009); and Letian et al., “Cellular Receptor Binding and Entry of Human Papillomavirus,” J. Virol. 7:2 (2010) which are hereby incorporated by reference in their entirety). Papillomaviruses, as most DNA viruses, have evolved slowly, and all infect the same target cell, the basal cell of the stratified epithelium. The epidemiologic uniformity of the infections and diseases caused by genital HPV does not argue for the evolution of different molecular mechanisms of entry. In fact, even HPV types that cause epidermodysplasia verruciformis do infect the genital epithelium (Potocnik et al., “Beta-Papillomaviruses in Anogenital Hairs Plucked from Healthy Individuals,” J. Med. Virol. 78(12):1673-1678 (2006)). If there are differences in the pathogenesis of the different HPV, they seem to be related to factors intervening after cell entry. Therefore, it is likely, that many, if not the majority of the differences noted in the entry mechanisms of various papillomaviruses, some very closely related like HPV-16 and -31, reflect the various artificial tools that have been used for this research. Native infectious HPV virions are rarely used, because they are difficult to obtain. As a consequence most of the research relies on the use of virus-like particles or pseudovirions. Furthermore, the research also relies on human keratinocyte monolayers or even cells that are not naturally infected by HPV, like mouse cells or epithelia.
Even though effective HPV vaccines are available, different strategies to prevent HPV transmission and infection still need to be addressed. Although a strategy like the use of a microbicide is cumbersome, as the product has to be applied prior to every sexual contact, and costly, several considerations argue for exploring the development of an effective microbicide for HPV, and more broadly, for other sexually transmitted agents. There are other factors that favor development of an effective microbicide for HPV and other sexually transmitted agents. The HPV vaccine coverage of the target population varies by country and is likely to shift, but in the US in 2007, only 25% of the adolescents aged 13 to 17 years had received at least one dose of the HPV vaccine Gardasil (Jain et al., “Vaccination Coverage Among Adolescents Aged 13-17 Years—United States, 2007,” MMWR 57:1100-1103 (2008)). Many barriers have been identified to increasing HPV vaccine coverage, and their disappearance is unlikely to be complete or sufficient to eliminate the need for additional preventative measures (Humiston et al., “Health Care Provider Attitudes and Practices Regarding Adolescent Immunizations: A Qualitative Study,” Patient Educ. Counsel 75(1):121-127 (2009)). Among the reasons given by young women for not receiving the vaccine, the most common answers were not being sexually active or not having time to get vaccinated (Jain et al., “Human Papillomavirus (HPV) Awareness and Vaccination Initiation Among Women in the United States, National Immunization Survey—Adult 2007,” Prev. Med. 48(5):426-431 (2009)). Should the sudden opportunity present to initiate sexual intercourse, these women are likely to seek, at least temporarily, an immediate, private, and easily accessible means of protection, which the vaccine is not. In addition, the HPV vaccine protects the vaccinee, not the sexual partner. A microbicide, in contrast, may be transferred during intercourse from the subject to his/her partner, protecting both individuals from the exchange of an unrecognized HPV infection.
The quest for an effective microbicide against sexually transmitted agents has been recognized to be of high priority by the National Institutes of Health. The only success thus far is a report for a recent study demonstrating that tenofovir in a gel vehicle was able to reduce HIV-1 incidence compared to placebo (Abdool Karim et al., “Effectiveness and Safety of Tenofovir Gel, an Antiretroviral Microbicide, for the Prevention of HIV Infection in Women,” Science 329:1168-1174 (2010)). Although this is the first report of a potential microbicide for a sexually transmitted disease, the antiviral agent tenofovir most likely acted as an early treatment of HIV-1 infection rather than as a virus entry blocking agent.
The current formulation of Gardasil offers to the HPV-naive individual complete protection against only four types of HPV—types 6, 11, 16, and 18-, and only partial protection against infection or cervical diseases caused by other non-vaccine HPV types (Brown et al., “The Impact of Quadrivalent Human Papillomavirus (HPV; types 6, 11, 16, and 18) L1 Virus-Like Particle Vaccine on Infection and Disease due to Oncogenic Nonvaccine HPV Types in Generally HPV—Naive Women aged 16-26 Years,” J. Infect. Dis. 199(7):926-935 (2009) and Wheeler et al., “The Impact of Quadrivalent Human Papillomavirus (HPV; types 6, 11, 16, and 18) L1 Virus-Like Particle Vaccine on Infection and Disease due to Oncogenic Nonvaccine HPV Types in Sexually Active Women Aged 16-26 Years,” J. Infect. Dis. 199(7):936-944 (2009)). Yet, these other non-vaccine types account for the majority of low grade cervical squamous intraepithelial lesions (SIL), and a substantial proportion of the high grade SIL (De Vuyst et al., “Prevalence and Type Distribution of Human Papillomavirus in Carcinoma and Intraepithelial Neoplasia of the Vulva, Vagina and Anus: A Meta-Analysis,” Internat. J. Cancer 124(7):1626-1636 (2009) and Clifford et al., “Chapter 3: HPV Type-Distribution in Women with and without Cervical Neoplastic Diseases,” Vaccine 24 Suppl. 3:S26-34 (2006)). The ability to block these infections would be a substantial health benefit. If the receptor(s) of viral entry is common to all genital HPV types, as it appears to be, then it would be desirable to identify a microbicide that could be sufficient to blockade the cellular entry of all HPV types.
It would be desirable, therefore, to provide a composition that is capable of blocking these receptors to prevent PV infection, preferably without risk of jeopardizing the efficacy of any vaccine, i.e., via inducing immune tolerance.
The present invention is directed to overcoming these and other deficiencies in the art.